人工锌指核酸酶突变EGFP基因的功能分析

锌指核酸酶技术在基因定点修饰中具有效率高和特异性好等特点，并成功应用于数十种生物。目前，该技术是否能应用羊上尚未报道。为了敲除转基因山羊标记基因 (EGFP)，构建了一对针对EGFP外显子上的锌指核酸酶表达载体，将其电转染至转EGFP基因胎儿成纤维细胞中，研究了锌指核酸酶突变EGFP基因的效率和方式，利用基因显微注射单细胞获得获得的转基因 (EGFP) 细胞系作为锌指核酸酶的靶细胞。结果显示，通过锌指核酸酶的突变作用，转染后的细胞发绿色荧光比例下降，测序结果显示在EGFP外显子中插入1个碱基G，导致编码EGFP基因的阅读框改变，从而起到基因突变的作用。结果表明，文中构建的锌指核酸酶对EGFP基因有突变作用，可以为以后获得无标记基因供核细胞进行体细胞核移植生产克隆羊奠定基础。

Targeted exogenous EGFP gene editing in caprine fetus fibroblasts by zinc-finger nucleases

Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.